STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.     

The heterogeneity of rat brain mitochondria isolated on continuous sucrose gradients

Abstract— The distribution of a series of enzymes in the post-nuclear supernatant of rat brain homogenates was investigated following continuous density-gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.     

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Release of the phophodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers.In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mictochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes.Activator was released from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles.The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

THE SUBCELLULAR DISTRIBUTION OF CARBONIC ANHYDRASE IN HOMOGENATES OF PERFUSED RAT BRAIN

Abstract— The distribution of carbonic anhydrase was examined in subcellular fractions of perfused rat brain and compared with those of markers for cytosol (lactic dehydrogenase), mitochondrial matrix (glutamic dehydrogenase), and mitochondrial membranes (succinic dehydrogenase). About half of the total carbonic anhydrase was found in particulate fractions, with the greatest part of this in the crude mitochondrial fraction. This fraction was separated into its components on a discontinuous sucrose gradient either as such or after isotonic mechanical disruption with a French pressure cell, and the resultant fractions were characterized by electron microscopy and by assay of marker enzymes.
Carbonic anhydrase was solubilized by mechanical disruption, but not to the same extent as lactic dehydrogenase. The highest specific activity for carbonic anhydrase was found in the myelin fraction of the gradient. A mitochondrial locus for carbonic anhydrase is unlikely, but the presence of the enzyme in synaptosomes remains in question.
Addition of soluble carbonic anhydrase did not significantly increase the activity of particulate fractions. Treatment of particulate fractions with detergent was necessary to reveal latent activity; this procedure resulted in a more than ten-fold increase in the measurable carbonic anhydrase activity of myelin fragments.     

Multiple disc gel bands of mitochondrial creatine and adenylate kinases

Abstract— The activities and electrophoretic patterns of creatine and adenylate kinases in the mitochondrial and high speed supernatant fractions of adult mouse brain were determined. Approximately 22 per cent of the activities of both kinases is firmly bound to the mitochondria. On acrylamide gel electrophoresis of creatine kinase, in addition to the major band previously described, there were several other bands found. Although present in both the mitochondrial and supernatant fractions these additional protein bands with creatine kinase activity were significantly more intense in the mitochondrial fraction. There was only onesecondary band of adenylate kinase activity in the mitochondrial fraction but additional bands were found in the soluble fraction.     

SUBCELLULAR LOCALIZATION OF [14C]ADENINE DERIVATIVES NEWLY-FORMED IN CEREBRAL TISSUES AND THE EFFECTS OF ELECTRICAL EXCITATION

Abstract— Guinea pig neocortical tissues were incubated with [¹⁴C]adenine, dispersed in cold isotonic sucrose and subcellular fractions prepared by centrifugation. Some 98 per cent of the assimilated ¹⁴C was found as acid-soluble nucleotides in the incubated tissues. In primary fractions obtained by differential centrifugation, about 60 per cent of the [¹⁴C]-nucleotides were in supernatant fractions, in distinction to ATP of which the greatest molar quantity (61 per cent of that in the dispersion) was in the crude mitochondrial fraction. When the crude mitochondrial fraction was separated by density gradient centrifugation, most ¹⁴C was found in synaptosomal fractions and about 85 per cent of this ¹⁴C was adenine nucleotides.
Electrical stimulation of incubating tissues immediately prior to their dispersion and centrifugation greatly diminished the proportion of ¹⁴C subsequently found in nucleotides (collectively) in the supernatant fraction, and increased their inosine and hypoxanthine. Stimulation increased the tissue's cyclic AMP but a preferential localization for this was not established. Results are tentatively interpreted in terms of liberation of an adenine derivative on excitation, and its action or reuptake at a tissue component different from that from which it was liberated. Fractionation of tissues which had been incubated with both [¹⁴C]-adenine and [³H]adenosine suggested that of the two compounds, more adenosine was taken up by synaptic regions in preference to other cellular regions of the tissue.     

ISOLATION OF AN INSULIN SECRETION GRANULE FRACTION

Goosefish islets were homogenized in 0.25 M sucrose and separated into nuclear, mitochondrial + secretion granule, microsomal, and supernatant fractions. Eighty per cent of the cytochrome oxidase activity and 75 per cent of the bioassayed insulin activity were found in the mitochondrial + secretion granule fraction (6000 g for 10 minutes). The mitochondrial + secretion granule fraction was further subfractionated by centrifugation (2 hours at 100,000 g and 0°C) using a continuous linear density gradient 1.0–2.0 M sucrose). Eighteen to 20 subfractions were collected by piercing the bottom of the tube and collecting drops. The total protein was distributed into a bimodal curve consisting of a high density component, which contained 90 per cent of the insulin (secretion granules), and a lower density component, which contained the cytochrome oxidase activity (mitochondria).     

SUBCELLULAR DISTRIBUTION OF SUBSTANCE P IN BOVINE HYPOTHALAMUS AND SUBSTANTIA NIGRA

Abstract— Subcellular distribution of substance P as measured by radioimmunoassay was studied in fractions and subfractions from bovine hypothalamus and substantia nigra. Most of the substance P was found in the crude mitochondrial fraction. Subfractionation of the crude mitochondrial fraction by density gradient centrifugation showed most of substance P was present in nerve ending particles. Subcellular particle integrity was assessed using enzyme markers.     

ISOLATION AND PARTIAL CHARACTERIZATION OF FRACTIONS ENRICHED IN SYNAPTOSOMES FROM CHICK BRAIN

–From a pool of hemispheres, optic lobes and cerebellum of chick 3 fractions containing synaptosomes have been prepared. They were obtained by subcellular fractionation of a homogenate and centrifugation of a crude mitochondrial suspension on a discontinuous Ficoll density gradient in iso-osmoticsucrose. The synaptosomal fractions were isolated from bands at the interface of 5–9, 9–12 and 12–16% Ficoll. The characterization of these fractions by marker enzymes, such as lactate dehydrogenase, acetyl-cholinesterase, monoamine oxidase, acid phosphatase and rotenone-sensitive and -insensitive NADH: cytochrome c reductase is reported. Electron microscopic analyses showed that the first fraction (AB) at the 5–9% Ficoll interface contained myelin and other membrane fragments as well as synaptosomes, the second fraction (C) at the 9–12% Ficoll interface contained mainly synaptosomes, and the third fraction (D) at the 12–16% Ficoll interface contained synaptosomes and free mitochondria. A fourth fraction (E) was obtained as a pellet, and was enriched in free mitochondria. There was fair agreement between the distribution pattern of the marker enzyme activities and the particles of the fractions seen by electron microscopy. The content of glycoprotein-bound N-acetylneuraminic acid and total phospholipid of these fractions has been determined. Relative to the mitochondrial fraction (E) the synaptosome fraction contained on basis of particulate protein, respectively, 2–3 times as much protein-bound N-acetylneuraminic acid and 10–20 per cent more total phospholipid.     

STUDIES ON ISOLATED CELL COMPONENTS : XVI. The Distribution of Acid Phenyl Phosphatase Activities in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge has been used to separate the major components of rat liver brei (soluble phase, ribosomes, microsomes, mitochondria, membranous fragments, and nuclei) during one centrifugation, by using a 1200 ml sucrose gradient varying linearly with radius from 17 to 55 per cent (w/w) with a "cushion" of 66 per cent sucrose at the rotor edge at speeds up to 30,000 RPM. Liver brei was found to contain a family of phosphatases (phenol disodium phosphate substrate, sodium malonate buffers and Turgitol NPX, a non-ionic detergent). Activity maxima at pH 4.1 and 5.9 were observed in untreated brei prepared in 0.25 M sucrose. The addition of the non-ionic detergent Turgitol NPX selectively caused the release of considerable additional activity between these optima. The activity measured at pH 4.1 was primarily associated with the cytoplasmic granules, while the activities at pH 4.8, 5.4 and 5.9 were found in both soluble phase and particulate-mitochondria and membranous fractions. The activities present beyond the region of the gradient occupied by the soluble phase (sample layer) were all bound to particles sedimentable at 105,536 g (average) in the preparative ultracentrifuge. The data suggest that the different activities are not similarly distributed between soluble phase and particulate fractions. When the data are expressed in terms of specific activity, the area in the gradient between the microsomes and mitochondria now appears richest in all the acid phenyl phosphatase activities measured, while the soluble phase and larger particulate fractions appear relatively poor in activity. This part of the gradient is occupied by small, dense granules which may be the so called lysosomes. Pretreatment of the brei with Turgitol NPX prior to fractionation in the zonal ultracentrifuge resulted in the solubilization of acid phenyl phosphatase activities (almost all the activity was in the sample zone of the gradient) and the non-specific destruction of the formed elements of the brei. Essentially all of the activities present in the original brei measured under these conditions were recovered after zonal ultracentrifuge fractionations.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.     

Subcellular distribution and properties of guanylate cyclase in rat cerebellum.

In rat cerebellum the major portion of guanylate cyclase was found to be particulate-bound. The properties of particulate and supernatant guanylate cyclases from the cerebellum were comparatively examined. Both enzymes required the same optimal concentration of Mn2+ and were stimulated by Ca2+ in the presence of a low concentration of Mn2+. But dispersion of the particulate enzyme with Triton X-100 altered the Mn2+ concentration producing maximum activity and the inhibitory effect of Ca2+. The subcellular distributions of guanylate and adenylate cyclases were also studied in rat cerebellum. The major portions of the two cyclases were found in the mitochondrial fraction. The submitochondrial fractions separated by sucrose gradient showed that the major activities of both cyclases were concentrated in the fraction containing mainly nerve ending particles.     

A Cytochemical Study on the Pancreas of the Guinea Pig : I. Isolation and Enzymatic Activities of Cell Fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.     

A cytochemical study on the pancreas of the guinea pig. I. Isolation and enzymatic activities of cell fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.     

The subsynaptosomal localization of histamine, histidine decarboxylase and histamine methyltransferase in rat hypothalamus

Abstract— We have examined the subcellular localization of histamine, histamine methyltransferase (EC 2.1.1.8) (HMT) and histidine decarboxylase (EC 4.1.1.22) in rat hypothalamus after osmotic lysis of synaptosome-containing primary particulate fractions. When crude mitochondrial fractions are subjected to osmotic lysis, histamine is retained within particulate structures, while HMT is released into the supernatant fluid. The majority of histidine decarboxylase activity is also recovered in the supernatant fluid, although more histidine decarboxylase than HMT is retained in particulate fractions. After sucrose gradient fractionation of osmotically lysed crude mitochondrial or microsomal pellets, histamine is also retained in particulate structures, with the greatest amount occurring in a fraction enriched in synaptic vesicles. In these sucrose gradients histidine decarboxylase activity shows a greater particulate localization than does HMT activity.     

Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat

Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.     

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.     

Isolation of pig platelet membranes and granules distribution and validity of marker enzymes

A method for the subcellular fractionation of pig platelet homogenates by sucrose density gradient centrifugation is described. The procedure is simple, highly reproducible and yields two major particulate fractions and a soluble phase. One particulate fraction consists almost entirely of membrane fragments and is relatively free from granule contamination. The other particulate zone contains the platelet granules and mitochondria. The distribution on the gradients of the enzymes lactate dehydrogenase, succinate dehydrogenase, 5′-nucleotidase, leucyl β-naphthylamidase and cholinesterase has been studied and organelle localisation further substantiated by electron microscopy. The degree of solubilisation of certain marker enzymes during homogenisation has been investigated and the parallel release of these enzymes with the soluble phase marker enzyme lactate dehydrogenase, suggests they have a true biphasic location between the soluble and particulate components of the cell. No significant difference was found in the molar ratios of cholesterol to phospholipid in the subcellular fractions but the content of each lipid was twice as high in the membrane fraction as in the granule fraction.